ORIGINAL RESEARCH article
Front. Plant Sci.
Sec. Functional and Applied Plant Genomics
Volume 16 - 2025 | doi: 10.3389/fpls.2025.1548766
Re-calibration of flow cytometry standards for plant genome size estimation
Provisionally accepted
- Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Queensland, Australia
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Flow cytometry (FCM) and genome sequencing are complementary methods for estimating plant genome size (GS). However, discrepancies between the GS estimates derived from genome assemblies and FCM create ambiguity regarding the accuracy of these approaches. Around 12,000 plant GS measurements have been reported, with hardly any of them based upon genome assemblies. Currently FCM is the most frequently used method. Accurate GS estimation by FCM relies on internal standards with known GS values. However, previous GS calibrations, often based on incomplete reference genome assemblies, have led to significant discrepancies in GS estimates. Historically, the GS of a diploid plant species was estimated by doubling the size of a consensus genome assembly. However, consensus assemblies collapse homologous chromosomes into a single sequence, typically favouring the larger haplotype and potentially overestimating GS especially in highly heterozygous species. Here, we have applied haplotype resolved genome assemblies to accurately recalibrate the reference standards. We utilized a recent gapless, telomere-to-telomere (T2T) consensus and the most complete phased genome assemblies of the Nipponbare rice as a primary standard to recalibrate five commonly used plant standards. Using the consensus genome as a reference revealed an overestimation of over 30% in widely used previous GS estimates for Pisum sativum and Nicotiana benthamiana, around 18% for Arabidopsis thaliana, and 5% for Sorghum bicolor, and Gossypium hirsutum. The GS estimates based on phased haplotype assemblies suggested an additional 6-7% overestimation. Haplotype-resolved genome assemblies allow recalibration of GS estimates with potential to yield more accurate values by capturing haplotype-specific variations previously missed in consensus assemblies.
Keywords: Flow Cytometry, Plant genome size, recalibration, genome assembly, Haplotypes, Nipponbare rice, Reference Standards, consensus genome assembly
Received: 20 Dec 2024; Accepted: 18 Sep 2025.
Copyright: © 2025 Soni and Henry. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Abhishek Soni, a.soni@uq.edu.au
Robert Henry, robert.henry@uq.edu.au
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